Evaluation of molecular apoptosis signaling pathways and its correlation with EBV viral load in SLE patients using systems biology approach

BACKGROUND: Considerable evidence supports that SLE could be related to apoptotic cells and EBV infection. OBJECTIVE: The aim of this study was to identify the transcriptional signature of EBV infection in SLE patients for survey of the molecular apoptosis signaling pathways. METHODS: The PBMCs gene expression profiles of healthy control and SLE patients were obtained from GEO. Functional annotation and signaling pathway enrichment were carried out using DAVID, KEGG. To validate bioinformatics analysis the changes in genes expression of some of obtained genes, Real time PCR was performed on PBMCs from 28 SLE patients and 18 controls. RESULTS: We found that mean viral load was 6013 ± 390.1 copy/μg DNA from PBMCs in all patients. QRT-PCR results showed that the expression of the DUSP1 and LAMP3 genes which had most changes in the logFC among 4 candidate genes, increased significantly in comparison with control. The consistent expression of LMP2 as viral latency gene involve in apoptosis signaling pathways was detected in SLE patients with EBV viral load and some controls. CONCLUSIONS: The study indicated that some cellular genes may have an important role in pathogenesis of SLE through apoptosis signaling pathways. Beside, EBV infection as an environmental risk factor for SLE may affect the dysfunction of apoptosis.

The Nerve Growth Factor IB-like Receptor Nurr1 (NR4A2) recruits CoREST transcription repressor complexes to silence HIV following proviral reactivation in microglial cells

Human immune deficiency virus (HIV) infection of microglial cells in the brain leads to chronic neuroinflammation, which is antecedent to the development of HIV-associated neurocognitive disorders (HAND) in the majority of patients. Productively HIV infected microglia release multiple neurotoxins including proinflammatory cytokines and HIV proteins such as envelope glycoprotein (gp120) and transactivator of transcription (Tat). However, powerful counteracting silencing mechanisms in microglial cells result in the rapid shutdown of HIV expression to limit neuronal damage. Here we investigated whether the Nerve Growth Factor IB-like nuclear receptor Nurr1 (NR4A2), which is a repressor of inflammation in the brain, acts to directly restrict HIV expression. HIV silencing was substantially enhanced by Nurr1 agonists in both immortalized human microglial cells ( hµglia ) and induced pluripotent stem cells (iPSC)-derived human microglial cells (iMG). Overexpression of Nurr1 led to viral suppression, whereas by contrast, knock down (KD) of endogenous Nurr1 blocked HIV silencing. Chromatin immunoprecipitation (ChIP) assays showed that Nurr1 mediates recruitment of the CoREST/HDAC1/G9a/EZH2 transcription repressor complex to HIV promoter resulting in epigenetic silencing of active HIV. Transcriptomic studies demonstrated that in addition to repressing HIV transcription, Nurr1 also downregulated numerous cellular genes involved in inflammation, cell cycle, and metabolism, thus promoting HIV latency and microglial homoeostasis. Thus, Nurr1 plays a pivotal role in modulating the cycles of proviral reactivation by cytokines and potentiating the proviral transcriptional shutdown. These data highlight the therapeutic potential of Nurr1 agonists for inducing HIV silencing and microglial homeostasis and amelioration of the neuroinflammation associated with HAND.

Comparative transcriptome analyses reveal genes associated with SARS-CoV-2 infection of human lung epithelial cells

During 2020, understanding the molecular mechanism of SARS-CoV-2 infection (the cause of COVID-19) became a scientific priority due to the devastating effects of the COVID-19. Many researchers have studied the effect of this viral infection on lung epithelial transcriptomes and deposited data in public repositories. Comprehensive analysis of such data could pave the way for development of efficient vaccines and effective drugs. In the current study, we obtained high-throughput gene expression data associated with human lung epithelial cells infected with respiratory viruses such as SARS-CoV-2, SARS, H1N1, avian influenza, rhinovirus and Dhori, then performed comparative transcriptome analysis to identify SARS-CoV-2 exclusive genes. The analysis yielded seven SARS-CoV-2 specific genes including CSF2 [GM-CSF] (colony-stimulating factor 2) and calcium-binding proteins (such as S100A8 and S100A9), which are known to be involved in respiratory diseases. The analyses showed that genes involved in inflammation are commonly altered by infection of SARS-CoV-2 and influenza viruses. Furthermore, results of protein–protein interaction analyses were consistent with a functional role of CSF2 and S100A9 in COVID-19 disease. In conclusion, our analysis revealed cellular genes associated with SARS-CoV-2 infection of the human lung epithelium; these are potential therapeutic targets.

Human cytomegalovirus RNA2.7 is required for upregulating multiple cellular genes to promote cell motility and viral spread late in lytic infection

Long non-coding RNAs are frequently associated with broad modulation of gene expression and thus provide the cell with the ability to synchronize entire metabolic processes. We used transcriptomic approaches to investigate whether the most abundant human cytomegalovirus-encoded lncRNA, RNA2.7, has this characteristic. By comparing cells infected with wild-type virus (WT) with cells infected with RNA2.7 deletion mutants, RNA2.7 was implicated in regulating a large number of cellular genes late in lytic infection. Pathway analysis indicated that >100 of these genes are associated with promoting cell movement, and the ten most highly regulated of these were validated in further experiments. Morphological analysis and live cell tracking of WT- and RNA2.7 mutant-infected cells indicated that RNA2.7 is involved in promoting the movement and detachment of infected cells late in infection, and plaque assays using sparse cell monolayers indicated that RNA2.7 is also involved in promoting cell-to-cell spread of virus. Consistent with the observation that upregulated mRNAs are relatively A+U-rich, which is a trait associated with transcript instability, and that they are also enriched in motifs associated with mRNA instability, transcriptional inhibition experiments on WT- and RNA2.7 mutant-infected cells showed that four upregulated transcripts were longer-lived in the presence of RNA2.7. These findings demonstrate that RNA2.7 is required for promoting cell movement and viral spread late in infection and suggest that this may be due to general stabilization of A+U-rich transcripts. IMPORTANCE In addition to messenger RNAs (mRNAs), the human genome encodes a large number of long non-coding RNAs (lncRNAs). Many lncRNAs that have been studied in detail are associated with broad modulation of gene expression and have important biological roles. Human cytomegalovirus, which is a large, clinically important DNA virus, specifies four lncRNAs, one of which (RNA2.7) is expressed at remarkably high levels during lytic infection. Our studies show that RNA2.7 is required for upregulating a large number of human genes, about 100 of which are associated with cell movement, and for promoting the movement of infected cells and the spread of virus from one cell to another. Further bioinformatic and experimental analyses indicated that RNA2.7 may exert these effects by stabilizing mRNAs that are relatively rich in A and U nucleotides. These findings increase our knowledge of how human cytomegalovirus regulates the infected cell to promote its own success.

Massive colonization of protein-coding exons by selfish genetic elements in Paramecium germline genomes

Ciliates are unicellular eukaryotes with both a germline genome and a somatic genome in the same cytoplasm. The somatic macronucleus (MAC), responsible for gene expression, is not sexually transmitted but develops from a copy of the germline micronucleus (MIC) at each sexual generation. In the MIC genome of Paramecium tetraurelia, genes are interrupted by tens of thousands of unique intervening sequences called internal eliminated sequences (IESs), which have to be precisely excised during the development of the new MAC to restore functional genes. To understand the evolutionary origin of this peculiar genomic architecture, we sequenced the MIC genomes of 9 Paramecium species (from approximately 100 Mb in Paramecium aurelia species to >1.5 Gb in Paramecium caudatum). We detected several waves of IES gains, both in ancestral and in more recent lineages. While the vast majority of IESs are single copy in present-day genomes, we identified several families of mobile IESs, including nonautonomous elements acquired via horizontal transfer, which generated tens to thousands of new copies. These observations provide the first direct evidence that transposable elements can account for the massive proliferation of IESs in Paramecium. The comparison of IESs of different evolutionary ages indicates that, over time, IESs shorten and diverge rapidly in sequence while they acquire features that allow them to be more efficiently excised. We nevertheless identified rare cases of IESs that are under strong purifying selection across the aurelia clade. The cases examined contain or overlap cellular genes that are inactivated by excision during development, suggesting conserved regulatory mechanisms. Similar to the evolution of introns in eukaryotes, the evolution of Paramecium IESs highlights the major role played by selfish genetic elements in shaping the complexity of genome architecture and gene expression.

MYC-Induced Replicative Stress: A Double-Edged Sword for Cancer Development and Treatment

MYC is a transcription factor that controls the expression of a large fraction of cellular genes linked to cell cycle progression, metabolism and differentiation. MYC deregulation in tumors leads to its pervasive genome-wide binding of both promoters and distal regulatory regions, associated with selective transcriptional control of a large fraction of cellular genes. This pairs with alterations of cell cycle control which drive anticipated S-phase entry and reshape the DNA-replication landscape. Under these circumstances, the fine tuning of DNA replication and transcription becomes critical and may pose an intrinsic liability in MYC-overexpressing cancer cells. Here, we will review the current understanding of how MYC controls DNA and RNA synthesis, discuss evidence of replicative and transcriptional stress induced by MYC and summarize preclinical data supporting the therapeutic potential of triggering replicative stress in MYC-driven tumors.

Stem Cell Theory of Cancer: Implications of a Viral Etiology in Certain Malignancies

In 1911, Peyton Rous (Nobel Prize winner in 1966) demonstrated that a virus (i.e., RSV) caused cancer in chickens. In 1976, Bishop and Varmus (Nobel Prize winners in 1989) showed that the cellular origin of retroviral oncogenes was actually normal cellular genes (i.e., proto-oncogenes). In this article, we revisit the role viruses play in the genetic origin of cancer. We review a link between viruses or cancer and autoimmunity in an alternative stem cell origin of cancer. We propose that a virus is more likely to cause cancer when it infects a progenitor stem-like cell rather than a progeny differentiated cell. We postulate that both known (e.g., HBV and HPV) and novel viruses (e.g., SARS-CoV-2) pose an imminent threat in the emergence of chronic viral diseases as well as virally induced malignancies. Knowing the origin of cancer has profound implications on our current conception and perception of cancer. It affects our conduct in cancer research and our delivery of cancer care. It would be ironic if viruses turn out to be a useful tool and an ideal means in our quest to verify a genetic versus stem cell origin of cancer. When it pertains, oncology recapitulates ontology; viral (or genetic) content may be king, but cellular (or stem-like) context is key.

Highly selective and potent p16-CDK-RB-E2F targeted oncolytic virus therapies.

e14543 Background: Growth factor and p16-CDK-RB-E2F pathway components are mutated in almost all cancers, resulting in oncogenic E2F transcriptional activity that drives uncontrolled proliferation. Chemotherapies indiscriminately kill all proliferating cells, resulting in limiting toxicities. CDK inhibitors are more targeted but cytostatic as opposed to tumoricidal. E2F was discovered as a factor that transcribes adenovirus (Ad) E2 and cellular genes to drive viral replication. Ad E1A binds to RB, which activates E2F. On this basis, viruses with E1A-RB binding mutations are being evaluated in the clinic as oncolytic viruses (OVs) that selectively replicate in and kill RB mutant tumor cells. However, E1A-RB mutations alone are not sufficient to prevent viral replication in normal cells. The addition of ‘tumor specific promoters’ improves tumor selectivity but drastically impacts replication and lytic activity. Therefore, a critical unmet need is the engineering of E2F addicted OVs that replicate like wildtype viruses in tumor cells, but leave normal cells unharmed. Methods: We have developed a proprietary platform that enables Ad therapeutics to be assembled from libraries of functional genomic parts that confer desirable properties. Here we describe E1 and E4 genomic modules that confer E2F dependent tumor selective replication. We assembled a panel of ̃50 viruses with combinations of mutations that ablate i) E1A interactions with RB/p107/p130 and/or EP300 ii) E4-ORF1 activation of PI3-Kinase and/or MYC iii) E4-ORF6/7 induced E2F1/DP1 dimerization. These viruses were screened in panels of primary, tumor, and CRISPR-engineered cells. Results: These studies reveal that E4-ORF6/7 functions independently of E1A to activate E2F transcriptional targets, S phase entry and viral replication. We show that viruses with both E1A and E4-ORF6/7 mutations (AdSyn-181) have the ideal tumor selectivity/efficacy profiles and outperform existing OVs. We also show that the deletion of p16 in primary cells rescues AdSyn-181 replication by ̃15 fold (̃20% wildtype virus), demonstrating the E2F dependence and tumor selectivity mechanism. Furthermore, we show that in tumor cells, which have multiple p16-CDK-RB pathway mutations, AdSyn-181 replication is rescued to 100% wildtype virus levels. ICVB-1042, which incorporates these selectivity mutations – together with other modules that enhance lytic cell death and spread, tumor tropisms and IV delivery – is in IND-enabling studies. Conclusions: Leveraging extensive research into the overlapping logic of viral and cancer pathways, we have engineered OVs, including ICVB-1042, that distinguish between tumor and normal cell proliferation. Furthermore, unlike therapies that target a single component, ICVB-1042 targets tumors with different p16-CDK-RB mutations, which all converge in activating oncogenic E2F transcription, driving exponential and tumoricidal viral replication.

Let it go: HIV-1 cis-acting repressive sequences

After human immunodeficiency virus type 1 (HIV-1) was identified in the early 1980s, intensive work began to understand the molecular basis of HIV-1 gene expression. Subgenomic HIV-1 RNA regions, spread throughout the viral genome, were described to have a negative impact on the nuclear export of some viral transcripts. These studies revealed an intrinsic RNA code as a new form of nuclear export regulation. Since such regulatory regions were later also identified in other viruses as well as in cellular genes, it can be assumed that during evolution, viruses took advantage of them to achieve more sophisticated replication mechanisms. Here, we review HIV-1 cis-acting repressive sequences that have been identified and discuss their possible underlying mechanisms and importance. Additionally, we show how current bioinformatic tools might allow more predictive approaches to identify and investigate them.

Vaccinia virus gene acquisition through non-homologous recombination

Many of the genes encoded by poxviruses are orthologs of cellular genes. These virus genes serve different purposes, but perhaps of most interest is the way some have been repurposed to inhibit the antiviral pathways that their cellular homologs still regulate. What is unclear is how these virus genes were acquired although it is presumed to have been catalyzed by some form(s) of non-homologous recombination (NHR). We used transfection assays and substrates encoding a fluorescent and drug selectable marker to examine the NHR frequency in vaccinia virus (VAC) infected cells. These studies showed that when cells were transfected with linear duplex DNAs bearing VAC N2L gene homology it yielded a recombinant frequency (RF) of 6.7×10−4. In contrast, DNA lacking any VAC homology reduced the yield of recombinants ∼400-fold (RF = 1.6×10−6). DNA·RNA hybrids were also substrates, although homologous molecules yielded fewer recombinants (RF = 2.1×10−5) and non-homologous substrates yielded only rare recombinants (RF ≤ 3×10−8). NHR was associated with genome rearrangements ranging from simple insertions with flanking sequence duplications to large-scale indels that produced helper-dependent viruses. The insert was often also partially duplicated and would rapidly rearrange through homologous recombination. Most of the virus-insert junctions exhibited little or no pre-exiting microhomology, although a few encoded VAC topoisomerase recognition sites (C/T·CCTT). These studies show that VAC can catalyze NHR through a process that may reflect a form of aberrant replication fork repair. Although it is less efficient than classical homologous recombination, the rates of NHR may still be high enough to drive virus evolution. IMPORTANCE Large DNA viruses sometimes interfere in antiviral defenses using repurposed and mutant forms of the cellular proteins that mediate these same reactions. Such virus orthologs of cellular genes were presumably captured through non-homologous recombination, perhaps in the distant past, but nothing is known about the processes that might promote “gene capture” or even how often these events occur over the course of an infectious cycle. This study shows that non-homologous recombination in vaccinia virus infected cells is frequent enough to seed a small but still significant portion of novel recombinants into large populations of newly replicated virus particles. This offers a route by which a pool of virus might survey the host genome for sequences that offer a selective growth advantage and potentially drive discontinuous virus evolution (saltation) through the acquisition of adventitious traits.